We have initiated studies to understand in detail how newly synthesized proteins which must be secreted from cells or inserted into biological membranes are identified and directed to transport sites in the endoplasmic reticulum (in eukaryotic cells) or the cytoplasmic membrane (in prokaryotic cells). The project combines biochemical and genetic approaches to investigate the role of the mammalian signal recognition particle (SRP), the signal recognition particle receptor (SRP receptor), and their bacterial homologs in this process. Previous studies have indicated that SRP and the SRP receptor are key components of the cellular machinery responsible for the "initiation" phase of protein export. We expect that this project will help to solve a number of important questions about two fundamental cellular processes, protein sorting and protein folding. In addition, aspects of this project will provide insight into the regulation of multi-step pathways and the function of proteins that have broad substrate specifies. A number of mutations have been introduced into a gene encoding the 54kd subunit of mammalian SRP (SRP54). This protein has been shown to recognize "signal sequences" that earmark proteins for entry into the endoplasmic reticulum. The mutations have been selected to alter either the signal sequence recognition properties of the protein or its GTPase activity. Analysis of the mutant proteins involving the use of established in vitro assays for signal sequence recognition and protein transport into the endoplasmic reticulum has been initiated. These experiments will help elucidate the mechanism whereby SRP54 recognizes a wide spectrum of signal sequence substrates and the exact role of its GTPase activity in regulating the access of proteins to the secretory pathway. Studies have also been initiated to understand the function of homologs of SRP and SRP receptor in E. coli. The ease of performing genetic experiments makes this organism attractive; moreover, studies of the function of SRP and the SRP receptor in an evolutionarily ancient species should yield important information about their function in all cells. We have initiated genetic experiments to firmly establish the role of these homologs in the protein export process. We have also begun to explore the possibility of establishing in vitro assays for protein export that are dependent upon the activity of the homologs. Finally, we are beginning to use biochemical methods to determine whether the SRP receptor homolog interacts with proteins that have previously been shown to play an important role in protein secretion.